1. The influence of a transmembrane (TM2) amino acid located at a homologous position in human β1 (X290) and β3 (N289) GABA(A) receptor subunits and the RDL GABA receptor of Drosophila (M314) upon allosteric regulation by general anaesthetics has been investigated. 2. GABA-evoked currents mediated by human wild-type (WT) α6β3γ(2L) or WT RDL GABA receptors expressed in Xenopus laevis oocytes were augmented by propofol or pentobarbitone. High concentrations of either anaesthetic directly activated α6β3γ(2L), but not RDL, receptors. 3. GABA-evoked currents mediated by human mutant GABA(A) receptors expressing the RDL methionine residue (i.e. α6β(3N289M)γ(2L)) were potentiated by propofol or pentobarbitone with ~ 2-fold reduced potency and, in the case of propofol, reduced maximal effect. Conspicuously, the mutant receptor was refractory to activation by either propofol or pentobarbitone. 4. Incorporation of the homologous GABA(A) β1-subunit residue in the RDL receptor (i.e. RDL(M314S)) increased the potency, but not the maximal effect, of GABA potentiation by either propofol or pentobarbitone. Strikingly, either anaesthetic now activated the receptor, an effect confirmed for propofol utilizing expression of WT or mutant RDL subunits in Schnieder S2 cells. At RDL receptors expressing the homologous β3-subunit residue (i.e. RDL(M314N)) the actions of propofol were similarly affected, whereas those of pentobarbitone were unaltered. 5. The results indicate that the identity of a homologous amino acid affects, in a complementary manner, the direct activation of human (α6β3γ(2L)) and RDL GABA receptors by structurally distinct general anaesthetics. Whether the crucial residue acts as a regulator of signal transduction or as a component of an anaesthetic binding site per se is discussed.